Chimeric Antigen Receptors
Traditionally, antigen-specific T-cells have been generated by selective expansion of peripheral blood T-cells natively specific for the target antigen. However, it is difficult and quite often impossible to select and expand large numbers of T-cells specific for most cancer antigens. Gene-therapy with integrating vectors affords us a solution to this problem: transgenic expression of Chimeric Antigen Receptor (CAR) allows large numbers of T-cells specific to any surface antigen to be easily generated by ex vivo viral vector transduction of a bulk population of peripheral blood T-cells.
The most common forms of these molecules are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies which recognise a target antigen, fused via a spacer and a transmembrane domain to a signalling endodomain. Such molecules result in activation of the T-cell in response to recognition by the scFv of its cognate target. When T cells express such a CAR, they recognize and kill target cells that express the target antigen. Several CARs have been developed against tumour associated antigens, and adoptive transfer approaches using such CAR-expressing T cells are currently in clinical trial for the treatment of various cancers. To-date however, the main clinical exploration and potential application of CAR therapy is as treatment for B-cell malignancies.
CARs Directed Against CD19
CD19 is a B-cell antigen which is expressed very early in B-cell differentiation and is only lost at terminal B-cell differentiation into plasma cells. Hence, CD19 is expressed on all B-cell malignancies apart from multiple myeloma. It is not expressed on other haematopoietic populations or non-haematopoietic cells and therefore targeting this antigen should not lead to toxicity to the bone marrow or non-haematopoietic organs. Loss of the normal B-cell compartment is considered an acceptable toxicity when treating lymphoid malignancies, because although effective CD19 CAR T cell therapy will result in B cell aplasia, the consequent hypogammaglobulinaemia can be treated with pooled immunoglobulin.
CD19 is therefore an attractive CAR target. To date, the main clinical focus of the CAR field has been studies targeting CD19 on refractory B-cell cancers, as summarised in Table 1.
Different designs of CARs have been tested against CD19 in different centres, as outlined in Table 1:
TABLE 1Summary of CAR experience targeting CD19CentreBinderEndodomainCommentUniversity CollegeFmc63CD3-ZetaLow-level briefLondonpersistenceMemorial SloaneSJ25C1CD28-ZetaShort-termKetteringpersistenceNCI/KITEFmc63CD28-ZetaLong-term low-levelpersistenceBaylor, Centre for CellFmc63CD3-Zeta/Short-term low-leveland Gene TherapyCD28-ZetapersistenceUPENN/NovartisFmc6341BB-ZetaLong-term high-levelpersistence
Most of the studies have tested CD19 CARs based on a scFv derived from the hybridoma fmc63. The most promising have been in the treatment of Acute Lymphoblastic Leukaemia (ALL).
Clinical Experience with CARs Against CD19
CD19 directed CAR therapy appears most effective in ALL. The first studies in ALL were published in Spring 2013, by groups from Memorial Sloane Kettering (Brentjens, et al. (2013) Leukemia. Sci. Transl. Med. 5, 177ra38) and the University of Pennsylvania. An update report of the latter study has recently been made (Maude et al. (2014) N. Engl. J. Med. 371, 1507-1517). Here, 25 patients under the age of 25 years and 5 over this age were treated. 90% achieved a complete response at one month, 22 of 28 evaluable cases achieved an MRD negative status and the 6 month event free survival rate was 67%. 15 patients received no further therapy after the study.
Brentjens et al., (as above) in the adult setting, treated 5 ALL patients (2 with refractory relapse, 2 with MRD positive disease and 1 who was MRD negative) with autologous T cells retrovirally transduced to express a CD19 CAR incorporating an scFv derived from the SJ25C1 hybridoma and a CD28 co-stimulatory domain. All of these achieved a deep molecular remission, enabling 4 of these patients to receive an allogeneic SCT. This precluded assessment of the durability of responses but CAR T cells were only detectable in the blood or bone marrow for 3-8 weeks after infusion. The patient who was not transplanted relapsed at 90 days with CD19+ disease. Subsequently, Davila et al. ((2014). Sci. Transl. Med. 6, 224ra25) have updated this cohort. 14 of 16 adult patients had detectable disease at the point of CAR T cell infusion, despite salvage chemotherapy and cyclophosphamide conditioning. 14 of 16 achieved a complete remission with or without count recovery including 7 of 9 patients with morphologic evidence of residual disease detectable after salvage chemotherapy. 12 of 16 patients achieved MRD negativity and this allowed 7 to undergo allogeneic transplantation by the time of publication. Responses were durable in some patients with 4 of 8 non-transplanted patients continuing in morphological remission at up to 24 months follow-up although the survival curves for this cohort are not yet stable.
A recently published study in a cohort of paediatric and young adult patients predominantly with ALL provides the first intention-to-treat analysis of its outcomes. This may help remove the bias inherent in excluding patients who do not receive the anticipated dose of CAR T cells (Lee et al. (2014) Lancet. doi:10.1016/50140-6736(14)61403-3). 21 patients were treated with a CD28 domain-containing second generation CAR. All but 2 patients received the anticipated T cell dose, highlighting the feasibility of delivering this treatment to those with refractory or multiply-relapsed ALL. This study shows the following efficacy: 67% achieving a complete remission and 60% of those with ALL achieving MRD negative status.
Immune Toxicity of CD19 CAR Therapy
Cytokine release syndrome (CRS) encompasses a range of inflammatory symptoms ranging from mild to multi-organ failure with hypotension and respiratory failure. Some degree of CRS occurs commonly in patients treated with CD19 CAR T cells.
Approximately 30% (21/73) patients treated in recent cohorts showed some degree of CRS (Davilia et al (2014) as above; Lee et al (2014) as above; Kochenderfer (2014) J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. doi:10.1200/JCO.2014.56.2025). CRS has also been seen in patients treated with blinatumomab, a bi-specific recombinant single-chain antibody recognising both CD19 and CD3. CRS typically occurs 5-21 days after CAR T cell infusion.
CRS can be life threatening and requires treatment in an intensive care setting. CRS is associated with elevated serum cytokine levels. The cytokines most significantly elevated are IL-6, IL-10 and interferon gamma (IFNγ). Clinical manifestations of severe CRS (fever, hepatosplenomegaly, coagulopathy and hyperferritinaemia) resemble macrophage activation syndrome (MAS) found for instance in patients with congenital defects in T-cells. This suggests that common immunopathological processes are involved. At present it is not clear which cell type (CAR T cells, dying tumour cells, or locally-activated macrophages) are responsible for production of the key cytokines, particularly IL-6. However, a key initiating factor in MAS is release of copious Interferon-gamma (López-Alvarez et al. (2009). Clin. Vaccine Immunol. CVI 16, 142-145).
Neurotoxicity
A number of patients in CD19 CAR studies across institutions have developed transient neurotoxicity with a spectrum of severity from aphasia to obtundation, delirium and seizures (Davilia et al (2014) as above). This appears to be restricted to patients with ALL and a similar syndrome has been documented after blinatumomab therapy. Brain imaging appears normal. Neurotoxicity may reflect high levels of systemic cytokines crossing the blood-brain barrier.
Persistence, Relapse and T-Cell Exhaustion
Durable responses appeared to correlate with higher peak levels of circulating CAR transduced T cells, as well as with the duration of B cell aplasia. With exception of patients relapsing with CD19− disease, relapse was generally associated with loss of circulating CAR T cells and recovery of normal B cells.
T cell exhaustion is a state of T cell dysfunction that arises during many chronic infections and cancer. It is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors. Recently, a clearer picture of the functional and phenotypic profile of exhausted T cells has emerged with expression of inhibitory receptor programmed death 1 (PD-1; also known as PDCD1), a negative regulator of activated T cells, being a key feature (Day et al. (2006) Nature 443, 350-354).
Responses in CD19 CAR studies suggest that persistence of T-cells for a protracted period at high levels seems to be important in effecting durable responses. A CD19 CAR which reduces T-cell exhaustion may result in improved clinical responses.
There is thus a need for an alternative CAR directed against CD19 which is not associated with the above disadvantages.